Prostaglandin H synthase: Resolved and unresolved mechanistic issues

The cyclooxygenase and peroxidase activities of prostaglandin H synthase (PGHS)-1 and -2 have complex kinetics, with the cyclooxygenase exhibiting feedback activation by product peroxide and irreversible self-inactivation, and the peroxidase undergoing an independent self-inactivation process. The mechanistic bases for these complex, non-linear steady-state kinetics have been gradually elucidated by a combination of structure/function, spectroscopic and transient kinetic analyses. It is now apparent that most aspects of PGHS-1 and -2 catalysis can be accounted for by a branched chain radical mechanism involving a classic heme-based peroxidase cycle and a radical-based cyclooxygenase cycle. The two cycles are linked by the Tyr385 radical, which originates from an oxidized peroxidase intermediate and begins the cyclooxygenase cycle by abstracting a hydrogen atom from the fatty acid substrate. Peroxidase cycle intermediates have been well characterized, and peroxidase self-inactivation has been kinetically linked to a damaging side reaction involving the oxyferryl heme oxidant in an intermediate that also contains the Tyr385 radical. The cyclooxygenase cycle intermediates are poorly characterized, with the exception of the Tyr385 radical and the initial arachidonate radical, which has a pentadiene structure involving C11-C15 of the fatty acid. Oxygen isotope effect studies suggest that formation of the arachidonate radical is reversible, a conclusion consistent with electron paramagnetic resonance spectroscopic observations, radical trapping by NO, and thermodynamic calculations, although moderate isotope selectivity was found for the H-abstraction step as well. Reaction with peroxide also produces an alternate radical at Tyr504 that is linked to cyclooxygenase activation efficiency and may serve as a reservoir of oxidizing equivalent. The interconversions among radicals on Tyr385, on Tyr504, and on arachidonate, and their relationships to regulation and inactivation of the cyclooxygenase, are still under active investigation for both PGHS isozymes.     

Phospholipid actions on PGHS-1 and -2 cyclooxygenase kinetics

Cyclooxygenase (COX) catalysis by prostaglandin H synthase (PGHS) is a key control step for regulation of prostanoid biosynthesis. Both PGHS isoforms are integral membrane proteins and their substrate fatty acids readily partition into membranes, but the impact of phospholipids and lipid membranes on COX catalysis and the actions of COX inhibitors are not well understood. We have characterized the COX kinetics and ibuprofen inhibition of the purified PGHS isoforms in the presence of phosphatidylcholine (PC) with varying acyl chain structure and physical state. PC was found to directly inhibit COX activity, with non-competitive inhibition by PC monomers binding away from the COX active site and competitive inhibition by micellar/bilayer forms of PC due to sequestration of the arachidonate substrate. Competitive inhibition by native membranes was observed in a comparison of COX kinetics in sheep seminal vesicle microsomes before and after solubilization of PGHS-1. PC liposomes significantly increase the inhibitory potency of ibuprofen against both PGHS isoforms without changing the reversible character of ibuprofen action or requiring binding of PGHS to the liposomes. These results suggest a useful conceptual framework for analyzing the complex interactions among the PGHS proteins, substrates, inhibitors and phospholipid.     

Role of Asn-382 and Thr-383 in activation and inactivation of human prostaglandin H synthase cyclooxygenase catalysis

Cyclooxygenase catalysis by prostaglandin H synthase-1 and -2 (PGHS-1 and -2) requires activation of the normally latent enzyme by peroxide-dependent generation of a free radical at Tyr-385 (PGHS-1 numbering) in the cyclooxygenase active site; the Tyr-385 radical has also been linked to self-inactivation processes that impose an ultimate limit on cyclooxygenase catalysis. Cyclooxygenase activation is more resistant to suppression by cytosolic glutathione peroxidase in PGHS-2 than in PGHS-1. This differential response to peroxide scavenging enzymes provides a basis for the differential catalytic regulation of the two PGHS isoforms observed in vivo. We sought to identify structural differences between the isoforms, which could account for the differential cyclooxygenase activation, and used site-directed mutagenesis of recombinant human PGHS-2 to focus on one heme-vicinity residue that diverges between the two isoforms, Thr-383, and an adjacent residue that is conserved between the isoforms, Asn-382. Substitutions of Thr-383 (histidine in most PGHS-1) with histidine or aspartate decreased cyclooxygenase activation efficiency by about 40%, with little effect on cyclooxygenase specific activity or self-inactivation. Substitutions of Asn-382 with alanine, aspartate, or leucine had little effect on the cyclooxygenase specific activity or activation efficiency but almost doubled the cyclooxygenase catalytic output before self-inactivation. Asn-382 and Thr-383 mutations did not appreciably alter the Km value for arachidonate, the cyclooxygenase product profile, or the Tyr-385 radical spectroscopic characteristics, confirming the structural integrity of the cyclooxygenase site. The side chain structures of Asn-382 and Thr-383 in PGHS-2 thus selectively influence two important aspects of cyclooxygenase catalytic regulation: activation by peroxide and self-inactivation.     

Distinct influences of carboxyl terminal segment structure on function in the two isoforms of prostaglandin H synthase

The cyclooxygenase activity of the two prostaglandin H synthase (PGHS) isoforms, PGHS-1 and -2, is a major control element in prostanoid biosynthesis. The two PGHS isoforms have 60% amino acid identity, with prominent differences near the C-terminus, where PGHS-2 has an additional 18-residue insert. Some mutations of the C-terminal residue in PGHS-1 and -2 have been found to disrupt catalytic activity and/or intracellular targeting of the proteins, but the relationship between C-terminal structure and function in the two isoforms has been poorly defined. Crystallographic data indicate the PGHS-1 and -2 C-termini are positioned to interact with the endoplasmic reticulum (ER) membrane, although the C-terminal segment structure was not resolved for either isoform. We constructed a series of C-terminal substitution, deletion, and insertion mutants of human PGHS-1 and -2 and evaluated the effects on cyclooxygenase activity and intracellular targeting in transfected COS-1 cells expressing the recombinant proteins. PGHS-1 cyclooxygenase activity was strongly disrupted by C-terminal substitutions and deletions, but not by elongation of the C-terminal segment, even when the ultimate residue was altered. Similar alterations to PGHS-2 had markedly less effect on cyclooxygenase activity. The results indicate that the functioning of the longer C-terminal segment in PGHS-2 is distinctly more tolerant of structural change than the shorter PGHS-1 C-terminal segment. C-Terminal substitutions or deletions did not change the subcellular localization of either isoform, even at short times after transfection, indicating that neither C-terminal segment contains indispensable intracellular targeting signals.     

Substitution of tyrosine for the proximal histidine ligand to the heme of prostaglandin endoperoxide synthase 2: implications for the mechanism of cyclooxygenase activation and catalysis

Prostaglandin H(2) synthesis by prostaglandin endoperoxide synthase (PGHS) requires the heme-dependent activation of the protein's cyclooxygenase activity. The PGHS heme participates in cyclooxygenase activation by accepting an electron from Tyr385 located in the cyclooxygenase active site. Two mechanisms have been proposed for the oxidation of Tyr385 by the heme iron: (1) ferric enzyme oxidizes a hydroperoxide activator and the incipient peroxyl radical oxidizes Tyr385, or (2) ferric enzyme reduces a hydroperoxide activator and the incipient ferryl-oxo heme oxidizes Tyr385. The participation of ferrous PGHS in cyclooxygenase activation was evaluated by determining the reduction potential of PGHS-2. Under all conditions tested, this potential (<-135 mV) was well below that required for reactions leading to cyclooxygenase activation. Substitution of the proximal heme ligand, His388, with tyrosine was used as a mechanistic probe of cyclooxygenase activation. His388Tyr PGHS-2, expressed in insect cells and purified to homogeneity, retained cyclooxygenase activity but its peroxidase activity was diminished more than 300-fold. Concordant with this poor peroxidase activity, an extensive lag in His388Tyr cyclooxygenase activity was observed. Addition of hydroperoxides resulted in a concentration-dependent decrease in lag time consistent with each peroxide's ability to act as a His388Tyr peroxidase substrate. However, hydroperoxide treatment had no effect on the maximal rate of arachidonate oxygenation. These data imply that the ferryl-oxo intermediates of peroxidase catalysis, but not the Fe(III)/Fe(II) couple of PGHS, are essential for cyclooxygenase activation. In addition, our findings are strongly supportive of a branched-chain mechanism of cyclooxygenase catalysis in which one activation event leads to many cyclooxygenase turnovers.     

Kinetic mechanism of the bifunctional enzyme prostaglandin-H-synthase. Effect of electron donors on the cyclooxygenase reaction

Prostaglandin-H-synthase (PGHS, EC 1.14.99.1) catalyzes the first committed step in biosynthesis of all prostaglandins, thromboxanes, and prostacyclins by converting arachidonic acid to prostaglandin H(2) (PGH(2)). PGHS exhibits two enzymatic activities: cyclooxygenase activity converting arachidonic acid to prostaglandin G(2) (PGG(2)) and peroxidase activity reducing the hydroperoxide PGG(2) to the corresponding alcohol, PGH(2). Despite the many investigations of the kinetics of PGHS, many features such as the absence of competition and mutual activation between the cyclooxygenase and peroxidase activities cannot be explained in terms of existing schemes. In this work we have studied the influence of different electron donors (N,N,N ,N -tetramethyl-p-phenylenediamine, L-epinephrine, 2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid), potassium ferrocyanide) on the PGHS activities. The proposed scheme describes independent but interconnected cyclooxygenase and peroxidase activities of PGHS. It also explains the experimental data obtained in the present work and known from the literature.     

Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid

Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O(2), and two electrons to prostaglandin endoperoxide H(2) (PGH(2)). Formation of PGH(2) involves an initial oxygenation of arachidonate to yield PGG(2) catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG(2) to form PGH(2) catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co(3+)-heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG(2) versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually make less but measurable contributions to optimal catalytic efficiency.     

Comparison of the properties of prostaglandin H synthase-1 and -2

Biosynthesis of prostanoid lipid signaling agents from arachidonic acid begins with prostaglandin H synthase (PGHS), a hemoprotein in the myeloperoxidase family. Vertebrates from humans to fish have two principal isoforms of PGHS, termed PGHS-1 and-2. These two isoforms are structurally quite similar, but they have very different pathophysiological roles and are regulated very differently at the level of catalysis. The focus of this review is on the structural and biochemical distinctions between PGHS-1 and-2, and how these differences relate to the functional divergence between the two isoforms.     

Molecular dynamics simulations of arachidonic acid complexes with COX-1 and COX-2: insights into equilibrium behavior

The cyclooxygenase (COX) enzymes are responsible for the committed step in prostaglandin biosynthesis, the generation of prostaglandin H(2). As a result, these enzymes are pharmacologically important targets for nonsteroidal antiinflammatory drugs, such as aspirin and newer COX-2 selective inhibitors. The cyclooxygenases are functional homodimers, and each subunit contains both a cyclooxygenase and a peroxidase active site. These enzymes are quite interesting mechanistically, as the conversion of arachidonic acid to prostaglandin H(2) requires two oxygenation and two cyclization reactions, resulting in the formation of five new chiral centers with nearly absolute regio- and stereochemical fidelity. We have used molecular dynamics (MD) simulations to investigate the equilibrium behavior of both COX-1 and COX-2 enzyme isoforms with bound arachidonate. These simulations were compared with reference simulations of arachidonate in solution to explore the effect of enzyme on substrate conformation and positioning in the active site. The simulations suggest that the substrate has greater conformational freedom in the COX-2 active site, consistent with the larger COX-2 active site volume observed in X-ray crystal structures. The simulations reveal different conformational behavior for arachidonate in each subunit over the course of extended equilibrium MD simulations. The simulations also provide detailed information for several protein channels that might be important for oxygen and water transport to or from active sites or for intermediate trafficking between the cyclooxygenase and peroxidase active sites. The detailed comparisons for COX-1 versus COX-2 active site structural fluctuations may also provide useful information for design of new isozyme-selective inhibitors.     

Prostaglandin biosynthesis can be triggered by lipid peroxides.

Studies of ferriheme cyclooxygenase, using two different assay systems, show that a variety of peroxides can trigger a rapid acceleration of cyclooxygenase activity to produce prostaglandins. Lipid hydroperoxides formed by lipoxygenase were the most potent activators tested, followed by prostaglandin G₂, which was slightly less potent. Peroxides nonspeciflcally generated during arachidonate autoxidation were as potent as the enzymatically formed lipid peroxides. These findings have important implications for cell function since any process which generates peroxides may activate the cyclooxygenase. Thus the balance between formation and removal of cellular lipid peroxides sets a peroxide tone that can regulate the rate of prostaglandin formation in cells.     

Comparison of the peroxidase reaction kinetics of prostaglandin H synthase-1 and -2.

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and -2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency. The kinetics of formation of Intermediate I (an Fe(IV) species with a porphyrin free radical) and Intermediate II (an Fe(IV) species with a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygenase catalysis) were monitored at 4 degrees c by stopped flow spectrophotometry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetraenoic acid, the rate constant for Intermediate I formation (k1) was 2.3 x 10(7) M-1 s-1 for PGHS-1 and 2.5 x 10(7) M-1 s-1 for PGHS-2, indicating that the isoforms have similar initial reactivity with this lipid hydroperoxide. For PGHS-1, the rate of conversion of Intermediate I to Intermediate II (k2) became the limiting factor when the hydroperoxide level was increased, indicating a rate constant of 10(2)-10(3) s-1 for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition between Intermediates I and II was not rate-limiting even at the highest hydroperoxide concentrations tested, indicating that the k2 value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that faster formation of the active cyclooxygenase species (Intermediate II) or increased stability of the active species increases the resistance of the cyclooxygenase to inhibition by the intracellular hydroperoxide scavenger, glutathione peroxidase. Kinetic differences between the PGHS isoforms in forming or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.     

Mechanical stimulation of skeletal muscle increases prostaglandin F2α production,cyclooxygenase activity,and cell growth by a pertussis toxin sensitive mechanism

Repetitive mechanical stimulation of differentiated skeletal muscle in tissue culture increased the long-term production of prostaglandin F_2α, an anabolic stimulator of myofiber growth. Within 4 h of initiating mechanical stimulation, the enzymatic activity of cyclooxygenase (prostaglandin GH synthase [PGHS]), a regulatory enzyme in prostaglandin synthesis, was increased 82% (P <.005), and this increase was maintained for at least 24 h. Kinetic analysis of stretch-activated cyclooxygenase activity indicated a two to threefold decrease in the enzyme's K_m, with little change in its V_max. Immunocytochemical analysis of the cell cultures indicated the presence of high levels of the mitogen-inducible isoform of cyclooxygenase (PGHS-2) in the skeletal myofibers compared to the interstitial fibroblasts. While the stretch-induced increase in cyclooxygenase enzymatic activity was not inhibited by tetrodotoxin and therefore was independent of cellular electrical activity, the G protein inhibitor pertussis toxin prevented stretch-induced cyclooxygenase activation. Pertussis toxin also inhibited stretch-induced increases in PGF_2α production, phospholipase D activation, and cell growth. It is concluded that stretch of skeletal muscle increases muscle cell growth through a G protein-dependent process involving the activation of cyclooxygenase, an immediate early gene product. © 1995 Wiley-Liss, Inc.     

Isolation of the cDNA for human prostaglandin H synthase

Prostaglandin H Synthase (PGHS, cyclooxygenase) is a 67 kd protein which catalyzes the first step in prostaglandin synthesis. The primary amino acid sequence and the molecular mechanisms regulating expression are unknown. We report here isolation of a cDNA clone for the enzyme from human vascular endothelial cells for use in such studies. High titre, polyclonal antiserum against PGHS was developed in rabbits. The antiserum was monospecific, reacted with cyclooxygenase on Western blots at a limiting dilution of 1:500,000 and immunoprecipitated cyclooxygenase synthesized by in vitro translation of PGHS messenger RNA. It was used to screen a lambda gt11 cDNA expression library from human endothelial cells. Three positive clones were isolated. Following plaque purification, one clone reacted strongly with two other polyclonal antisera independently raised against highly purified cyclooxygenase and the aspirin-acetylated enzyme. Western blot analysis confirmed production of a large approximately 180 kd fusion protein of cyclooxygenase and beta-galactosidase. The cDNA insert of approximately 2.2 kilo base pairs was excised and subcloned into plasmid pUC8. A 24 nucleotide DNA probe, synthesized according to the amino acid sequence of the aspirin-acetylation site of cyclooxygenase, hybridized strongly with the 2.2 kbp cDNA insert. It is concluded that the 2.2 kbp cDNA insert represents a cDNA clone for human cyclooxygenase, which also expresses the aspirin-acetylation site. This is the first reported isolation of the cDNA for this enzyme, and will facilitate further studies on the primary sequence and on the regulation of the enzyme at the molecular level.     

Antiproliferative activity of guava leaf extract via inhibition of prostaglandin endoperoxide H synthase isoforms

Prostaglandin endoperoxide H synthase (PGHS) is a key enzyme for the synthesis of prostaglandins (PGs) which play important roles in inflammation and carcinogenesis. Because the extract from Psidium guajava is known to have a variety of beneficial effects on our body including the anti-inflammatory, antioxidative and antiproliferative activities, we investigated whether the extract inhibited the catalytic activity of the two PGHS isoforms using linoleic acid as an alternative substrate. The guava leaf extract inhibited the cyclooxygenase reaction of recombinant human PGHS-1 and PGHS-2 as assessed by conversion of linoleic acid to 9- and 13-hydroxyoctadecadienoic acids (HODEs). The guava leaf extract also inhibited the PG hydroperoxidase activity of PGHS-1, which was not affected by nonsteroidal anti-inflammatory drugs (NSAIDs). Quercetin which was one of the major components not only inhibited the cyclooxygenase activity of both isoforms but also partially inhibited the PG hydroperoxidase activity. Overexpression of human PGHS-1 and PGHS-2 in the human colon carcinoma cells increased the DNA synthesis rate as compared with mock-transfected cells which did not express any isoforms. The guava leaf extract not only inhibited the PGE₂ synthesis but also suppressed the DNA synthesis rate in the PGHS-1- and PGHS-2-expressing cells to the same level as mock-transfected cells. These results demonstrate the antiproliferative activity of the guava leaf extract which is at least in part caused by inhibition of the catalytic activity of PGHS isoforms.     

Structural and catalytic insights into the algal prostaglandin H synthase reveal atypical features of the first non-animal cyclooxygenase

Prostaglandin H synthases (PGHSs) have been identified in the majority of vertebrate and invertebrate animals, and most recently in the red alga Gracilaria vermiculophylla. Here we report on the cloning, expression and characterization of the algal PGHS, which shares only about 20% of the amino acid sequence identity with its animal counterparts, yet catalyzes the conversion of arachidonic acid into prostaglandin-endoperoxides, PGG₂ and PGH₂. The algal PGHS lacks structural elements identified in all known animal PGHSs, such as epidermal growth factor-like domain and helix B in the membrane binding domain. The key residues of animal PGHS, like catalytic Tyr-385 and heme liganding His-388 are conserved in the algal enzyme. However, the amino acid residues shown to be important for substrate binding and coordination, and the target residues for nonsteroidal anti-inflammatory drugs (Arg-120, Tyr-355, and Ser-530) are not found at the appropriate positions in the algal sequences. Differently from animal PGHSs the G. vermiculophylla PGHS easily expresses in Escherichia coli as a fully functional enzyme. The recombinant protein was identified as an oligomeric (evidently tetrameric) ferric heme protein. The preferred substrate for the algal PGHS is arachidonic acid with cyclooxygenase reaction rate remarkably higher than values reported for mammalian PGHS isoforms. Similarly to animal PGHS-2, the algal enzyme is capable of metabolizing ester and amide derivatives of arachidonic acid to corresponding prostaglandin products. Algal PGHS is not inhibited by non-steroidal anti-inflammatory drugs. A single copy of intron-free gene encoding for PGHS was identified in the red algae G. vermiculophylla and Coccotylus truncatus genomes.     

Cytoplasmic prostaglandin E2 synthase is dominantly expressed in cultured KAT-50 thyrocytes,cells that express constitutive prostaglandin-endoperoxide H synthase-2. Basis for low protaglandin E2 production

The recent identification and cloning of two glutathione-dependent prostaglandin E(2) synthase (PGES) genes has yielded important insights into the terminal step of PGE(2) synthesis. These enzymes form efficient functional pairs with specific members of the prostaglandin-endoperoxide H synthase (PGHS) family. Microsomal PGES (mPGES) is inducible and works more efficiently with PGHS-2, the inflammatory cyclooxygenase, while the cytoplasmic isoform (cPGES) pairs functionally with PGHS-1, the cyclooxygenase that ordinarily exhibits constitutive expression. KAT-50, a well differentiated thyroid epithelial cell line, expresses high levels of PGHS-2 but surprisingly low levels of PGE(2) when compared with human orbital fibroblasts. Moreover, PGHS-1 protein cannot be detected in KAT-50. We report here that KAT-50 cells express high basal levels of cPGES but mPGES mRNA and protein are undetectable. Thus, KAT-50 cells express the inefficient PGHS-2/cPGES pair, and this results in modest PGE(2) production. The high levels of cPGES and the absence of mPGES expression result from dramatic differences in the activities of their respective gene promoters. When mPGES is expressed in KAT-50 by transiently transfecting the cells, PGE(2) production is up-regulated substantially. These observations indicate that naturally occurring cells can express a suboptimal profile of PGHS and PGES isoforms, resulting in diminished levels of PGE(2) generation.     

Prostaglandin H synthase: distinct binding sites for cyclooxygenase and peroxidase substrates

Prostaglandin H synthase has two distinct catalytic activities: a cyclooxygenase activity that forms prostaglandin G2 from arachidonic acid; and a peroxidase activity that reduces prostaglandin G2 to prostaglandin H2. Lipid hydroperoxides, such as prostaglandin G2, also initiate the cyclooxygenase reaction, probably via peroxidase reaction cycle enzyme intermediates. The relation between the binding sites for lipid substrates of the two activities was investigated with an analysis of the effects of arachidonic and docosahexaenoic acids on the reaction kinetics of the peroxidase activity, and their effects on the ability of a lipid hydroperoxide to initiate the cyclooxygenase reaction. The cyclooxygenase activity of pure ovine synthase was found to have an apparent Km value for arachidonate of 5.3 microM and a Ki value (competetive inhibitor) for docosahexaenoate of 5.2 microM. When present at 20 microM neither fatty acid had a significant effect on the apparent Km value of the peroxidase for 15-hydroperoxyeicosatetraenoic acid: the values were 7.6 microM in the absence of docosahexaenoic acid and 5.9 microM in its presence, and (using aspirin-treated synthase) 13.7 microM in the absence of arachidonic acid and 15.7 microM in its presence. Over a range of 1 to 110 microM the level of arachidonate had no significant effect on the initiation of the cyclooxygenase reaction by 15-hydroperoxyeicosatetraenoic acid. The inability of either arachidonic acid or docosahexaenoic acid to interfere with the interaction between the peroxidase and lipid hydroperoxides indicates that the cyclooxygenase and peroxidase activities of prostaglandin H synthase have distinct binding sites for their lipid substrates.     

Autocatalytic tyrosine nitration of prostaglandin endoperoxide synthase-2 in LPS-stimulated RAW 264.7 macrophages

In the literature, biological tyrosine nitrations have been reported to depend not only on peroxynitrite but also on nitrite/hydrogen peroxide linked to catalysis by myeloperoxidase. In endotoxin-stimulated RAW 264.7 macrophages, we have detected a major nitrotyrosine positive protein band around 72 kDa and identified it as prostaglandin endoperoxide synthase-2 (PGHS-2). Isolated PGHS-2 in absence of its substrate arachidonate was not only tyrosine-nitrated with peroxynitrite, but also with nitrite/hydrogen peroxide in complete absence of myeloperoxidase. Our data favor an autocatalytic activation of nitrite by PGHS-2 with a subsequent nitration of the essential tyrosine residue in the cyclooxygenase domain. Under inflammatory conditions, nitrite formed via NO-synthase-2 may therefore act as an endogenous regulator for PGHS-2 in stimulated macrophages. Nitration of PGHS-2 by the autocatalytic activation of nitrite further depends on the intracellular concentration of arachidonate since arachidonate reacted competitively with nitrite and could prevent PGHS-2 from nitration when excessively present.     

Peroxide-induced radical formation at TYR385 and TYR504 in human PGHS-1

Prostaglandin H synthase isoforms 1 and -2 (PGHS-1 and -2) react with peroxide to form a radical on Tyr385 that initiates the cyclooxygenase catalysis. The tyrosyl radical EPR signals of PGHS-1 and -2 change over time and are altered by cyclooxygenase inhibitor binding. We characterized the tyrosyl radical dynamics using wild type human PGHS-1 (hPGHS-1) and its Y504F, Y385F, and Y385F/Y504F mutants to determine whether the radical EPR signal changes involve Tyr504 radical formation, Tyr385 radical phenyl ring rotation, or both. Reaction of hPGHS-1 with peroxide produced a wide singlet, whereas its Y504F mutant produced only a wide doublet signal, assigned to the Tyr385 radical. The cyclooxygenase specific activity and K_M value for arachidonate of hPGHS-1 were not affected by the Y504F mutation, but the peroxidase specific activity and the K_M value for peroxide were increased. The Y385F and Y385F/Y504F mutants retained only a small fraction of the peroxidase activity; the former had a much-reduced yield of peroxide-induced radical and the latter essentially none. After binding of indomethacin, a cyclooxygenase inhibitor, hPGHS-1 produced a narrow singlet but the Y504F mutant did not form a tyrosyl radical. These results indicate that peroxide-induced radicals form on Tyr385 and Tyr504 of hPGHS-1, with radical primarily on Tyr504 in the wild type protein; indomethacin binding prevented radical formation on Tyr385 but allowed radical formation on Tyr504. Thus, hPGHS-1 and -2 have different distributions of peroxide-derived radical between Tyr385 and Tyr504. Y504F mutants in both hPGHS-1 and -2 significantly decreased the cyclooxygenase activation efficiency, indicating that formation of the Tyr504 radical is functionally important for both isoforms.